Congenital cardiac defects affect nearly 1% of all live births and are the most common cause of infant mortality in the United States. Among the most severe forms of congenital heart defects are those involved in outflow tract and interventricular septation, whereas valve anomalies are the most common form of congenital heart defect. The long-term goal of the proposed studies is to define the molecular mechanisms and pathways that control cardiovascular development and underscore these congenital cardiac anomalies. Specifically, this application is focused on the identification of transcriptional pathways and mechanisms that function during the development of the second heart field (SHF) and its derivatives in the OFT and RV. The LIM-homeodomain transcription factor Isl1 and the MADS domain transcription factor MEF2C are essential regulators of SHF development. Recent work has established that the Mef2c gene is a direct transcriptional target of Isl1 in the SHF, forming the basis of a transcriptional pathway in the AHF. However, the upstream transcriptional regulators of the Isl1 gene in the SHF remain undefined in spite of the early, essential role for Isl1 in that lineage. Similarly, the downstream targets of MEF2C are not known, even though Mef2c is required for heart development, and its function is required specifically in the SHF for proper OFT alignment and endocardial cushion remodeling. This proposal will therefore test the hypothesis that MEF2C is required in the SHF to control cell number or migration of SHF-derived cells into the OFT endocardial cushions and that MEF2C is a direct transcriptional activator of Nfatc1 in the endocardial cushions. This is significant since the transcription factor NFATc1 is required for endocardial cushion remodeling and heart valve maturation. This proposal will also test the hypothesis that the expression of the Isl1 gene in the SHF is regulated via a novel modular enhancer that integrates upstream signals to direct expression in cardiovascular progenitors in the anterior lateral mesoderm. Two specific aims are proposed. Specific aim one will determine the requirement of MEF2C in OFT alignment and endocardial cushion development by examining the requirement of Mef2c for migration of SHF-derived cells into the heart and for cell proliferation and survival using a conditional knockout approach in mice. Specific Aim 1 will also determine if Nfatc1 is a direct transcriptional target of MEF2C in the outflow tract endocardial cushions via a conserved, consensus MEF2 site present in the Nfatc1 endocardial- specific enhancer using a transgenic mouse approach. Specific Aim 2 will identify regulators of Isl1 transcription in the SHF by defining upstream regulators of a novel SHF-specific Isl1 transcriptional enhancer. The goal is to place Isl1 into a transcriptional and signaling pathway, which will provide essential information about the early events controlling heart development and Isl1+ progenitor self-renewal and differentiation.